PROJECTSUMMARY Brain ischemia is a leading cause of morbidity and mortality in the United States. We seek to develop therapeutics to reduce the extent of damage and functional impairment resulting from ischemic injury to the brain, an area of significant unmet medical need. In a mouse model of stroke injury we have demonstrated thatthesyntheticdsRNA,polyinosinic-polycytidylicacidstabilizedbypoly-L-lysineandcarboxymethylcellulose (PIC, Hiltonol), is a robust prophylactic neuroprotectant against ischemic injury. PIC given to mice one day prior to transient middle cerebral artery occlusion reduced the area of damage in the brain by ~95%. We recentlypublishedthatinterferon(IFN)receptorsignalingisrequiredforPIC-inducedneuroprotectionagainst mousestroke,providingapotentialmechanisticbiomarkerforpredictingneuroprotectivedoses.Basedonour studies in mice, we hypothesize that preconditioning is mediated through systemic IFN secretion and consequentinductionofinterferonregulatedgenesinthebrainandthatthesemarkerscanbeusedtoidentify efficaciousdosesfortranslationtonon-humanprimates(NHP)studiesandultimatelyhumanclinicaltrials. R21SpecificAims: Aim1.ValidatebiomarkersinrhesusmacaquesthatbestreflectPICbioactivityinducedbyefficacious doses in the mouse. Milestone Define a dose of PIC that results in the induction of the mechanistic biomarkerIFN?andatleast3ofthe4othercytokinebiomarkersintheNHPtolevelswithintheinterquartile range(25th-75thpercentile)offoldchangesseeninthemouseatefficaciousdoses.Ifwefailtoidentifyadose thatmeetsbothconditionswewillrelysolelyoninductionofthemechanisticbiomarkerIFN?.R21Criteriafor Success. The primary goal is to establish a neuroprotective dose across species based on the induction of systemicbiomarkersassociatedwithefficaciousdoses.Ano-godecisionwillbereachedifwefailtoidentifya doseofPICthatresultinanincreaseofthemechanisticbiomarker,IFN?. R33SpecificAim: Aim1.EvaluatethepotentialofPICtoprotectagainstcerebralischemicinjuryinNHPs.Milestone1) Establish a dose that demonstrates a >25% reduction in neurological score in a majority of animals preconditionedwithPICand/or>25%reductionininfarctvolume(T2magneticresonanceimages;?day2).We viewthismilestoneasessentialforprogressionofPICtoclinicalpreconditioningstudies.2)Establishaclinical monitoring strategy of biomarkers that correlate with improved outcomes. A biomarker strategy would greatly reducetheriskoffailureinclinicalstudiesbyprovidingameanstobetterestablishtargetdosesinpopulations withsignificantco-morbidities.